Protein-based genome editing for vegetable crops without genetic modification

Overview

This solution addresses a critical barrier in vegetable crop breeding by enabling genome editing without producing genetically modified organisms. Current genome editing methods rely on plant transformation and the creation of a T0 genetically modified generation, which raises societal acceptance concerns. The proposed approach uses pre-assembled CAS9/gRNA ribonucleoproteins delivered directly into plant cells through two complementary methods: microinjection into partially digested cells and biolistic delivery using a Crystallized Liposome Acceleration System (CLAS). By delivering editing machinery as functional protein complexes rather than DNA, this approach bypasses the production of transgenic intermediate plants, potentially accelerating regulatory approval and market adoption of edited vegetable varieties.

Technical specifications

Key features:

• Partial cell wall digestion reduces cell wall thickness without producing complete protoplasts, facilitating protein insertion while preserving cell integrity for faster recovery and tissue regeneration
• Microinjection of pre-assembled ribonucleoproteins into partially digested plant cells enables direct delivery of CRISPR editing machinery
• Crystallized Liposome Acceleration System (CLAS) uses solid crystallised liposomes as carriers strong enough for biolistic delivery of ribonucleoproteins into plant cells
• Marker-free recovery eliminates the need for selectable markers during tissue regeneration
• In vivo validated CRISPR ribonucleoprotein activity demonstrated in prior gene editing experiments on target genes in vegetable crops

Technical approach:

The method leverages two validated capabilities: partial cell wall digestion protocols developed at the host institution and crystallised liposome technology currently undergoing intellectual property protection. The Plant Genetic Engineering Laboratory at the University of Queensland provides expertise in ribonucleoprotein-based gene editing across multiple vegetable crops.

Technology readiness level

This technology is currently at an early-to-mid development stage. The underlying techniques—partial cell digestion and crystallised liposome technology—have been demonstrated in laboratory settings, and CRISPR ribonucleoproteins have been validated for gene editing activity in vivo. The Crystallised Liposome technology is in the process of obtaining intellectual property protection. Future validation steps include optimising CAS9/gRNA protein concentrations, refining partial digestion parameters for microinjection, evaluating editing efficiency in microinjection experiments, optimising liposome crystallisation with ribonucleoproteins, calibrating biolistic bombardment parameters, and assessing chromosomal inversions and recovered plant efficiency in target vegetable crops. Collaborative partnerships with research institutions experienced in vegetable crop gene editing will support advancement toward practical breeding applications.